Detection of analytes using reorganization energy

ABSTRACT

The invention relates to novel methods and compositions for the detection of analytes using the nuclear reorganization energy, λ, of an electron transfer process.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/832,792,filed Aug. 2, 2007, which is a continuation of application Ser. No.11/283,233, filed Nov. 18, 2005, now U.S. Pat. No. 7,267,939, which is acontinuation of application Ser. No. 09/841,809, filed Apr. 24, 2001,now U.S. Pat. No. 7,018,523, which is a continuation of application Ser.No. 09/417,988, filed Oct. 13, 1999, now U.S. Pat. No. 6,248,229, whichis a continuation of application Ser. No. 09/096,504, filed Jun. 12,1998, now U.S. Pat. No. 6,013,170, which is a continuation ofapplication Ser. No. 08/873,977, filed Jun. 12, 1997, now U.S. Pat. No.6,013,459, all which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to novel methods and compositions for thedetection of analytes based on changes in the nuclear reorganizationenergy, λ, of electron transfer process.

BACKGROUND OF THE INVENTION

Electron transfer reactions are crucial steps in a variety of biologicaltransformations ranging from photosynthesis to aerobic respiration.Studies of electron transfer reactions in both chemical and biologicalsystems have led to the development of a large body of knowledge and astrong theoretical base, which describes the rate of electron transferin terms of a definable set of parameters.

Electronic tunneling in proteins and other biological molecules occursin reactions where the electronic interaction of the redox centers isrelatively weak. Semiclassical theory reaction predicts that thereaction rate for electron transfer depends on the driving force (−ΔG°),a nuclear reorganization parameter (λ), and the electronic-couplingstrength (H_(AB)) between the reactants and products at the transitionstate, according to the following equation:

k _(ET)=(4π³ /h ² λk _(B) T)^(1/2)(H _(AB))²exp[(−ΔG°+λ)² /λk _(B) T]

The nuclear reorginzation energy, λ, in the equation above is defined asthe energy of the reactants at the equilibrium nuclear configuration ofthe products. There are two components of λ; “outer sphere” effects(λ_(o)) and “inner sphere” effects (λ_(i)). For electron transferreactions in polar solvents, the dominant contribution to λ arises fromthe reorientation of solvent molecules in response to the change incharge distribution of the reactants. The second component of λ comesfrom the changes in bond lengths and angies due to changes in theoxidation state of the donors and acceptors.

It is an object of the present invention to provide methods for thedetection of target analytes exploiting changes in the solventreorganization energy of electron transfer reactions.

SUMMARY OF THE INVENTION

In accordance with the above objects, the present invention providesmethods of detecting a target analyte in a test sample. The methodcomprises binding an analyte to a redox active complex. The redox activecomplex comprises a solvent accessible transition metal complex havingat least one coordination site occupied by a polar coordination groupand a binding ligand which will bind the target analyte. The complex isbound to an electrode. Upon binding, a solvent inhibited transitionmetal complex is formed and electron transfer is detected between thesolvent inhibited transition metal complex and the electrode. Themethods also include applying at least a first input signal to thesolvent inhibited transition metal complex.

In a further aspect, the invention provides methods of detecting atarget analyte in a test sample comprising associating an analyte with aredox active complex. The redox active complex comprises a solventinhibited transition metal complex, and a binding ligand which will bindthe target analyte. Upon association, a solvent accessible transitionmetal complex is formed, which is then detected.

In an additional aspect, the invention provides methods of detecting atarget analyte in a test sample comprising associating an analyte with aredox active complex. The complex comprises a solvent inhibitedtransition metal complex, a binding ligand which will bind the targetanalyte, and an analyte analog. The complex is bound to an electrode,and upon association, a solvent accessible transition metal complex isformed, which is then detected.

In a further aspect, the invention provides compositions comprising anelectrode with a covalently attached redox active complex. The complexcomprises a binding ligand and a solvent accessible redox activemolecule, which has at least one, and preferably two or threecoordination sites occupied by a polar coordination group, one or moreof which may be a water molecule.

In a further aspect, the present invention provides an apparatus for thedetection of target analytes in a test sample, comprising a test chambercomprising a first and a second measuring electrode. The first measuringelectrode comprises a covalently attached redox active complexcomprising a solvent accessible transition metal complex, preferablyhaving at least three coordination sites occupied by a polarcoordination group, and a binding ligand. The apparatus furthercomprises an AC/DC voltage source electrically connected to the testchamber, and an optional signal processor for detection.

DETAILED DESCRIPTION

The present invention provides methods and compositions for thedetection of target analytes using changes in the solvent reorganizationenergy of transition metal complexes upon binding of the analytes, tofacilitate electron transfer between the transition metal complex and anelectrode. This invention is based on the fact that a change in theoxidation state of a redox active molecule such as a transition metalion, i.e. upon the acceptance or donation of an electron, results in achange in the charge and size of the metal ion. This change in thecharge and size requires that the surrounding solvent reorganize, tovarying degrees, upon this change in the oxidation state.

For the purposes of this invention, the solvent reorganization energywill be treated as the dominating component of λ. Thus, if the solventreorganization energy is high, a change in the oxidation state will beimpeded, even under otherwise favorable conditions.

In conventional methodologies using electron transfer, this solventeffect is minimized by using transition metal complexes that minimizesolvent reorganization at the redox center, generally by using severallarge hydrophobic ligands which serve to exclude water. Thus, the ligandfor the transition metal ions traditionally used are non-polar and aregenerally hydrophobic, frequently containing organic rings.

However, the present invention relies on the novel idea of exploitingthis solvent reorganization energy to serve as the basis of an assay fortarget analytes. In the present invention, transition metal complexesthat are solvent accessible, i.e. have at least one, and preferablymore, small, polar ligands, and thus high solvent reorganizationenergies, are used. Thus, at initiation energies less than the solventreorganization energy, no significant electron transfer occurs. However,upon binding of a generally large target analyte, the transition metalcomplexes becomes solvent inhibited, inaccessible to polar solventsgenerally through steric effects, which allows electron transfer atpreviously inoperative initiation energies.

Thus, the change in a transition metal complex from solvent accessibleto solvent inhibited serves as a switch or trigger for electrontransfer. This thus becomes the basis of an assay for an analyte. Clossand Miller have shown that there is a decrease in lambda in nonpolarsolvents in their work on Donor(bridge)Acceptor electron transferreactions in solution. (Closs and Miller, Science, 240, 440-447, (1988).This idea also finds conceptual basis in work done with metmyoglobin,which contains a coordinated water molecule in the hexacoordinate hemeiron site and does not undergo self-exchange very rapidly (rate constantk₂₂ 1M⁻¹s⁻¹). Upon chemical modification, the heme becomespentacoordinate, removing the water, and the self-exchange rate constantincreases significantly (rate constant k₂₂ 1×10⁴ M⁻¹s⁻¹); see Tsukahara,J. Am. Chem. Soc. 111:2040 (1989).

Without being bound by theory, there are two general mechanisms whichmay be exploited in the present invention. In a preferred embodiment,the binding of a target analyte to a binding ligand which is stericallyclose to a solvent accessible transition metal complex causes one ormore of the small, polar ligands on the solvent accessible transitionmetal complex to be replaced by one or more coordination atoms suppliedby the target analyte, causing a decrease in the solvent reorganizationenergy for at least two reasons. First, the exchange of a small, polarligand for a generally larger, nonpolar ligand that will generallyexclude more water from the metal, lowering the required solventreorganization energy (i.e. an inner sphere λ_(i) effect). Secondly, theproximity of a generally large target analyte to the relatively smallredox active molecule will sterically exclude water within the first orsecond coordination sphere of the metal ion, also decreasing the solventreorganization energy.

Alternatively, a preferred embodiment does not necessarily require theexchange of the polar ligands on the metal ion by a target analytecoordination atom. Rather, in this embodiment, the polar ligands areeffectively irreversibly bound to the metal ion, and the decrease insolvent reorganization energy is obtained as a result of the exclusionof water in the first or second coordination sphere of the metal ion asa result of the binding of the target analyte; essentially the water isexcluded (i.e. an outher sphere λ_(o) effect).

Accordingly, the present invention provides methods for the detection oftarget analytes. The methods generally comprise binding an analyte to abinding ligand that is either associated with (forming a redox activecomplex) or near to a transition metal complex. The transition metalcomplex is bound to an electrode generally through the use of aconductive oligomer. Upon analyte binding, the reorganization energy ofthe transition metal complex decreases to form a solvent inhibitedtransition metal complex, to allow greater electron transfer between thesolvent inhibited transition metal complex and the electrode.

Accordingly, the present invention provides methods for the detection oftarget analytes. By “target analyte” or “analyte” or grammaticalequivalents herein is meant any molecule, compound or particle to bedetected. As outlined below, target analytes preferably bind to bindingligands, as is more fully described below.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eucaryotic cells, including mammalian tumorcells); viruses (including retroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc. Particularly preferred analytesare environmental pollutants; nucleic acids; proteins (includingenzymes, antibodies, antigens, growth factors, cytokines, etc);therapeutic and abused drugs; cells; and viruses.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, a nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate, phosphorodithioate,O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc.114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992);Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996),all of which are incorporated by reference). Nucleic acids containingone or more carbocyclic sugars are also included within the definitionof nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176).These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of moieties, or to increase the stability andhalf-life of such molecules in physiological environments.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathanine andhypoxathanine, etc. As used herein, the term “nucleoside” includesnucleotides, and modified nucleosides such as amino or thio modifiednucleosides.

By “proteins” or grammatical equivalents herein is meant proteins,oligopeptides and peptides, and analogs, including proteins containingnon-naturally occurring amino acids and amino acid analogs, andpeptidomimetic structures.

As will be appreciated by those in the art, a large number of analytesmay be detected using the present methods; basically, any target analytefor which a binding ligand, described below, may be made may be detectedusing the methods of the invention.

In a preferred embodiment, the target analyte is added or introduced toa redox active complex, which is preferably attached to an electrode. By“redox active complex” herein is meant a complex comprising at least onetransition metal complex and at least one binding ligand, which, as morefully described below, may be associated in a number of different ways.By “transition metal complex” or “redox active molecule” or “electrontransfer moiety” herein is meant a metal-containing compound which iscapable of reversibly or semi-reversibly transferring one or moreelectrons. It is to be understood that electron donor and acceptorcapabilities are relative; that is, a molecule which can lose anelectron under certain experimental conditions will be able to accept anelectron under different experimental conditions. It is to be understoodthat the number of possible transition metal complexes is very large,and that one skilled in the art of electron transfer compounds will beable to utilize a number of compounds in the present invention.Transition metals are those whose atoms have a partial or complete dshell of electrons. Suitable transition metals for use in the inventioninclude, but are not limited to, cadmium (Cd), copper (Cu), cobalt (Co),palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh),osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc), titanium (Ti),Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni), Molybdenum(Mo), technetium (Tc), tungsten (W), and iridium (Ir). That is, thefirst series of transition metals, the platinum metals (Ru, Rh, Pd, Os,Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred. Particularlypreferred are metals that do not change the number of coordination sitesupon a change in oxidation state, including ruthenium, osmium, iron,platinium and palladium, with ruthenium and iron being especiallypreferred. Generally, transition metals are depicted herein as M.

The transition metal ions are complexed with ligands that serve toprovide the coordination atoms for the binding of the metal ion.Generally, it is the composition or characteristics of the ligands thatdetermine whether a transition metal complex is solvent accessible. By“solvent accessible transition metal complex” or grammatical equivalentsherein is meant a transition metal complex that has at least one,preferably two, and more preferably three, four or more small polarligands. The actual number of polar ligands will depend on thecoordination number (n) of the metal ion. Preferred numbers of polarligands are (n−1) and (n−2). For example, for hexacoordinate metals,such as Fe, Ru, and Os, solvent accessible transition metal complexespreferably have one to five small polar ligands, with two to five beingpreferred, and three to five being particularly preferred, depending onthe requirement for the other sites, as is more fully described below.Tetracoordinate metals such as Pt and Pd preferably have one, two orthree small polar ligands.

It should be understood that “solvent accessible” and “solventinhibited” are relative terms. That is, at high applied energy, even asolvent accessible transition metal complex may be induced to transferan electron.

The other coordination sites of the metal are used for attachment of thetransition metal complex to either a binding ligand (directly orindirectly using a linker), to form a redox active complex, or to theelectrode (frequently using a spacer, as is more fully described below),or both. Thus for example, when the transition metal complex is directlyjoined to a binding ligand, one, two or more of the coordination sitesof the metal ion may be occupied by coordination atoms supplied by thebinding ligand (or by the linker, if indirectly joined). In addition, oralternatively, one or more of the coordination sites of the metal ionmay be occupied by a spacer used to attach the transition metal complexto the electrode. For example, when the transition metal complex isattached to the electrode separately from the binding ligand as is morefully described below, all of the coordination sites of the metal (n)except 1 (n−1) may contain polar ligands.

Suitable small polar ligands, generally depicted herein as “L”, fallinto two general categories, as is more fully described below. In oneembodiment, the small polar ligands will be effectively irreversiblybound to the metal ion, due to their characteristics as generally poorleaving groups or as good sigma donors, and the identity of the metal.These ligands may be referred to as “substitutionally inert”.Alternatively, as is more fully described below, the small polar ligandsmay be reversibly bound to the metal ion, such that upon binding of atarget analyte, the analyte may provide one or more coordination atomsfor the metal, effectively replacing the small polar ligands, due totheir good leaving group properties or poor sigma donor properties.These ligands may be referred to as “substitutionally labile”. Theligands preferably form dipoles, since this will contribute to a highsolvent reorganization energy.

Irreversible ligand groups include, but are not limited to, amines(—NH₂, —NHR, and —NR₂, with R being a substitution group that ispreferably small and hydrophilic, as will be appreciated by those in theart), cyano groups (—C≡N), thiocyano groups (—SC≡N), and isothiocyanogroups (—N≡CS). Reversible ligand groups include, but are not limitedto, H₂O and halide atoms or groups. It should be understood that thechange in solvent reorganization energy is quite high when a watermolecule serves as a coordination atom; thus, the replacement oraddition of a single water molecule on a redox active molecule willgenerally result in a detectable change, even when the other ligands arenot small polar ligands. Thus, in a preferred embodiment, the inventionrelies on the replacement or addition of at least one water molecule ona redox active molecule.

In addition to small polar ligands, the metal ions may have additional,hydrophobic ligands, also depicted herein as “L”. That is, ahexacoordinate metal ion such as Fe may have one ligand position(preferably axial) filled by the spacer used for attachment to theelectrode, two ligand positions filled by phenanthroline, and two orthree small polar ligands, depending on the linkage to the bindingligand. As will be appreciated by those in the art, a wide variety ofsuitable ligands may be used. Suitable traditional ligands include, butare not limited to, isonicotinamide; imidazole; bipyridine andsubstituted derivatives of bipyridine; terpyridine and substitutedderivatives; phenanthrolines, particularly 1,10-phenanthroline(abbreviated phen) and substituted derivatives of phenanthrolines suchas 4,7-dimethylphenanthroline and dipyridol[3,2-a:2′,3′-c]phenazine(abbreviated dppz); dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene(abbreviated hat); 9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,1-tetra-azacyclotetradecane (abbreviated cyclam), isocyanide andmetallocene ligands. Substituted derivatives, including fusedderivatives, may also be used.

The presence of at least one small, polar ligand on the transition metalcomplex makes the solvent reorganization energy high, which suppresseselectron transfer to and from the transition metal redox activemolecule. Thus, in a preferred embodiment, a solvent accessible redoxactive molecule has a solvent reorganization energy of greater thanabout 500 meV, with greater than about 800 meV being preferred, greaterthan about 1 eV being especially preferred and greater than about 1.2 to1.3 eV being particularly preferred.

In addition to the solvent accessible redox active molecule, a redoxactive complex comprises a binding ligand which will bind the targetanalyte. By “binding ligand” or grammatical equivalents herein is meanta compound that is used to probe for the presence of the target analyte,and that will specifically bind to the analyte; the binding ligand ispart of a binding pair. By “specifically bind” herein is meant that theligand binds the analyte, with specificity sufficient to differentiatebetween the analyte and other components or contaminants of the testsample. This binding should be sufficient to remain bound under theconditions of the assay, including wash steps to remove non-specificbinding. Generally, the disassociation constants of the analyte to thebinding ligand will be in the range of at least 10⁴-10⁻⁶ M⁻¹, with apreferred range being 10⁻⁵ to 10⁻⁹ M⁻¹ and a particularly preferredrange being 10⁻⁷-10⁻⁹ M⁻¹.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand may be a complementarynucleic acid. Alternatively, the binding ligand may be a nucleicacid-binding protein when the analyte is a single or double-strandednucleic acid. When the analyte is a protein, the binding ligands includeproteins or small molecules. Preferred binding ligand proteins includepeptides. For example, when the analyte is an enzyme, suitable bindingligands include substrates and inhibitors. Antigen-antibody pairs,receptor-ligands, and carbohydrates and their binding partners are alsosuitable analyte-binding ligand pairs.

In general, preferred embodiments utilize relatively small bindingligands and larger target analytes.

Together, the transition metal complex and the binding ligand comprise aredox active complex. In addition, there may be more than one bindingligand or transition metal complex per redox active complex. The redoxactive complex may also contain additional moieties, such ascross-linking agents, labels, etc., and linkers for attachment to theelectrode.

The redox active complex is bound to an electrode. This may beaccomplished in any number of ways, as will be apparent to those in theart. Generally, as is more fully described below, one or both of thetransition metal complex and the binding ligand are attached, via aspacer, to the electrode.

In a preferred embodiment, the redox active complex is covalentlyattached to the electrode via a spacer. By “spacer” herein is meant amoiety which holds the redox active complex off the surface of theelectrode. In a preferred embodiment, the spacer is a conductiveoligomer as described herein, although suitable spacer moieties includepassivation agents and insulators as outlined below. The spacer moietiesmay be substantially non-conductive, although preferably (but notrequired) is that the electron coupling between the redox activemolecule and the electrode (H_(AB)) does not become the rate limitingstep in electron transfer.

In general, the length of the spacer is as described for conductivepolymers and passivation agents. As will be appreciated by those in theart, if the spacer becomes too long, the electronic coupling between theredox active molecule and the electrode will decrease.

In a preferred embodiment, the spacer is a conductive oligomer. By“conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. Conductive oligomers, and theirsynthesis, use and attachment to moieties is described in PCTUS97/20014, hereby expressly incorporated in its entirety.

By “substantially conducting” herein is meant that the electron couplingbetween the transition metal complex and the electrode (H_(AB)) throughtthe oligomer is not the rate limiting step of electron transfer.Generally, the conductive oligomer has substantially overlappingπ-orbitals, i.e. conjugated π-orbitals, as between the monomeric unitsof the conductive oligomer, although the conductive oligomer may alsocontain one or more sigma (a) bonds. Additionally, a conductive oligomermay be defined functionally by its ability to pass electrons into orfrom an attached transition metal complex. Furthermore, the conductiveoligomer is more conductive than the insulators as defined herein.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10⁴Ω⁻¹ cm⁻¹, with from about 10⁻⁵to about 10³Ω⁻¹ cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20Δ to about 200Δ. Asdescribed below, insulators have a conductivity S of about 10⁻⁷Ω⁻¹ cm⁻¹or lower, with less than about 10⁻⁸Ω⁻¹ cm⁻¹ being preferred. Seegenerally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during synthesis of theredox active complexes, ii) during the attachment of the conductiveoligomer to an electrode, or iii) during analyte assays.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to transition metalcomplexes or redox active complexes, binding ligands, electrodes, etc.or to several of these. Unless otherwise noted, the conductive oligomersdepicted herein will be attached at the left side to an electrode; thatis, as depicted in Structure 1, the left “Y” is connected to theelectrode as described herein and the right “Y”, if present, is attachedto the redox active complex, i.e. either the transition metal complex orbinding ligand, either directly or through the use of a linker, as isdescribed herein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B-D is a bond able to conjugate with neighboringbonds (herein referred to as a “conjugated bond”), preferably selectedfrom acetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR—and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen or phosphorus. Thus, suitable heteroatommoieties include, but are not limited to, —NH and —NR, wherein R is asdefined herein; substituted sulfur; sulfonyl (—SO₂—) sulfoxide (—SO—);phosphine oxide (—PO— and —RPO—); and thiophosphine (—PS— and —RPS—).However, when the conductive oligomer is to be attached to a goldelectrode, as outlined below, sulfur derivatives are not preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise an oligomer of a single type of Ygroups, or of multiple types of Y groups. Thus, in a preferredembodiment, when a barrier monolayer is used as is described below, oneor more types of Y groups are used in the conductive oligomer within themonolayer with a second type(s) of Y group used above the monolayerlevel. Thus, as is described herein, the conductive oligomer maycomprise Y groups that have good packing efficiency within the monolayerat the electrode surface, and a second type(s) of Y groups with greaterflexibility and hydrophilicity above the monolayer level to facilitatetarget analyte binding. For example, unsubstituted benzyl rings maycomprise the Y rings for monolayer packing, and substituted benzyl ringsmay be used above the monolayer. Alternatively, heterocylic rings,either substituted or unsubstituted, may be used above the monolayer.Additionally, in one embodiment, heterooligomers are used even when theconductive oligomer does not extend out of the monolayer.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. when the conductiveoligomers form a monolayer on the electrode, R groups may be used toalter the association of the oligomers in the monolayer. R groups mayalso be added to 1) alter the solubility of the oligomer or ofcompositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,ketones, iminos, sulfonyl, silicon moieties, halogens, sulfur containingmoieties, phosphorus containing moieties, and ethylene glycols. In thestructures depicted herein, R is hydrogen when the position isunsubstituted. It should be noted that some positions may allow twosubstitution groups, R and R′, in which case the R and R′ groups may beeither the same or different.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur, silicon or phosphorus. Alkyl alsoincludes heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, andsilicone being preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms including but not limited to, thia-, thio- and sulfo-compounds, thiols (—SH and —SR), sulfides (—RSR—), sulfoxides(—R—SO—R—), sulfones (—R—SO₂—R—), disulfides (—R—S—S—R—) and sulfonylester (R—SO₂—O—R) groups. By “phosphorus containing moieties” herein ismeant compounds containing phosphorus, including, but not limited to,phosphines and phosphates. By “silicon containing moieties” herein ismeant compounds containing silicon, including siloxanes.

By “ether” herein is meant an —O—R group.

By “ester” herein is meant a —COOR group; esters include thioesters(—CSOR).

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCOH groups.

By “ketone” herein is meant —R—CO—R groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “imino” herein is meant and —R—CNH—R— and —R—CNR—R— groups.

By “ethylene glycol” herein is meant a —(O—CH₂—CH₂)_(n)— group, althougheach carbon atom of the ethylene group may also be singly or doublysubstituted, i.e. —(O—CR₂CR₂)_(n)—, with R as described above. Ethyleneglycol derivatives with other heteroatoms in place of oxygen (i.e.—(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or with substitution groups) arealso preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, and ethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B-D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B-Dis a conjugated bond, containing overlapping or conjugated n-orbitals.

Preferred B-D bonds are selected from acetylene (—C═C—, also calledalkyne or ethyne), alkene (—CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B-D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 1 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B-Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B-D bond may bean amide bond, and the rest of the B-D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B-D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B-D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, to give greaterflexibility for nucleic acid hybridization.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient binding of target analytes, thebinding should occur at a distance from the surface. For example, itappears that the kinetics of nucleic acid hybridization increase as afunction of the distance from the surface, particularly for longoligonucleotides of 200 to 300 basepairs. Accordingly, the length of theconductive oligomer is such that the binding ligand is positioned fromabout 6Δ to about 100Δ (although distances of up to 500Δ may be used)from the electrode surface, with from about 25Δ to about 60Δ beingpreferred. Accordingly, n will depend on the size of the aromatic group,but generally will be from about 1 to about 20, with from about 2 toabout 15 being preferred and from about 3 to about 10 being especiallypreferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B-D bond or D moiety,i.e. the D atom is attached to the redox active complex or molecule, orbinding ligand, either directly or via a linker. In some embodimentsthere may be additional atoms, such as a linker, attached between theconductive oligomer and the bound moiety. Alternatively, when m is 1,the conductive oligomer may terminate in Y, an aromatic group, i.e. thearomatic group is attached to the moiety or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 4 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. See forexample, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361 (1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour, Chem.Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below.

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B-D is acetyleneand Y is phenyl or substituted phenyl. A preferred embodiment ofStructure 2 is also when e is one, depicted as Structure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B-D is azo; Y is phenyl or substituted phenyl and B-D isalkene; Y is pyridine or substituted pyridine and B-D is acetylene; Y isthiophene or substituted thiophene and B-D is acetylene; Y is furan orsubstituted furan and B-D is acetylene; Y is thiophene or furan (orsubstituted thiophene or furan) and B-D are alternating alkene andacetylene bonds.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 4:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofnitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide, and G is abond selected from alkane, alkene or acetylene, such that together withthe two carbon atoms the C-G-C group is an alkene (—CH═CH—), substitutedalkene (—CR═CR—) or mixtures thereof (—CH═CR— or —CR═CH—), acetylene(—C≡C—), or alkane (—CR₂—CR₂—, with R being either hydrogen or asubstitution group as described herein). The G bond of each subunit maybe the same or different than the G bonds of other subunits; that is,alternating oligomers of alkene and acetylene bonds could be used, etc.However, when G is an alkane bond, the number of alkane bonds in theoligomer should be kept to a minimum, with about six or less sigma bondsper conductive oligomer being preferred. Alkene bonds are preferred, andare generally depicted herein, although alkane and acetylene bonds maybe substituted in any structure or embodiment described herein as willbe appreciated by those in the art.

In a preferred embodiment, the m of Structure 4 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 5:

The alkene oligomer of structure 5, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of Structures 1 and 4.

The conductive oligomers are covalently attached to the redox activecomplexes, transition metal complexes (collectively redox activemoieties), or binding ligands. By “covalently attached” herein is meantthat two moieties are attached by at least one bond, including sigmabonds, pi bonds and coordination bonds.

The redox active moiety or binding ligand is covalently attached to theconductive oligomer, and the conductive oligomer is also covalentlyattached to the electrode. In general, the covalent attachments are donein such a manner as to minimize the amount of unconjugated sigma bondsan electron must travel from the electron donor to the electronacceptor. Thus, linkers are generally short, or contain conjugated bondswith few sigma bonds.

The covalent attachment of the redox active moiety or binding ligand andthe conductive oligomer may be accomplished in a variety of ways, andwill depend on the composition of the redox active moiety or bindingligand, as will be appreciated by those in the art. Representativeconformations of the attachment of redox active complexes to electrodesare depicted below in Structures 6 and 7:

In Structure 6, the hatched marks on the left represent an electrode. Xis a conductive oligomer as defined herein. F₁ is a linkage that allowsthe covalent attachment of the electrode and the conductive oligomer,including bonds, atoms or linkers such as are described herein. F₂ is alinkage that allows the covalent attachment of the conductive oligomerto the redox active complex, which includes the binding ligand, BL. F₁and F₂ may be a bond, an atom or a linkage as is herein described. F₂may be part of the conductive oligomer, part of the redox activecomplex, or exogeneous to both. As for Structure 7, M is the metal ionand L is a co-ligand, as defined herein; as noted above, if atraditional hydrophobic ligand is used, two or more of the depicted Lligands may be part of multidentate ligand, rather than separateligands. It should be noted that while the BL is depicted in an axialposition, this is not required.

In this embodiment, a coordination atom may be contributed directly fromthe binding ligand; alternatively, there may be a linker that provides acoordination atom and is linked to the binding ligand. A wide variety oflinkers can be used herein, as will be appreciated by those in the art.Suitable linkers are known in the art, for example, homo- orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). Preferred linkers include, but arenot limited to, alkyl groups and alkyl groups containing heteroatommoieties, with short alkyl groups, esters, epoxy groups and ethyleneglycol and derivatives being preferred, with propyl, acetylene, and C₂alkene being especially preferred.

Structure 7 depicts a “branched” conformation, wherein the transitionmetal complex and the binding ligand are not directly attached. As willbe appreciated by those in the art, the transition metal complex and thebinding ligand may be attached at the same position on the conductiveoligomer, or different positions, and more than one transition metalcomplex and/or binding ligand may be present.

Structures 6 and 7 depict hexacoordinate metal ions, although as will beappreciated by those in the art, other types of metal ions also find usein the invention, with the appropriate adjustment of L ligands.

The attachment of the metal ion is generally done by attaching asubstitutionally inert ligand to the end of the spacer. In a preferredembodiment, this ligand is monodentate, or at most bidentate, althoughother polydentate ligands may also be used. Thus, for example, an aminoor imidazole group (monodentate) or a phenathroline (bidentate) may beattached to the end of the spacer using techniques well known in theart, or techniques outlined in PCT US97/20014, hereby expresslyincorporated by reference.

The attachment of the binding ligand to either the metal ion or thespacer is also done using well known techniques, and will depend on thecomposition of the binding ligand. When the binding ligand is a nucleicacid, either double-stranded or single-stranded, attachment to the metalion can be done as is described in PCT US97/20014.

In general, attachment of the binding ligand to either the metal ion orthe spacer is done using functional groups either naturally found on thebinding ligand or added using well known techniques. These groups can beat the terminus of the binding ligand, for example at the N- orC-terminus of a protein, or at any internal position. Thus, amino, thio,carboxyl or amido groups can all be used for attachment. Similarly,chemical attachment of traditional ligands such as pyridine orphenanthroline may also be done, as will be appreciated by those in theart. For example, attachment of proteinaceous binding ligands isgenerally done using functional groups present on the amino acid sidechains or at the N- or C-terminus; for example, any groups such as theN-terminus or side chains such as histidine may serve as ligands for themetal ion. Similarly, attachment of carbohydrate binding ligands isgenerally done by derivatizing the sugar to serve as a metal ion ligand.Alternatively, these groups may be used to attach to the spacer, usingwell known techniques. In any of these embodiments, there may beadditional connector or linkers present. For example, when the bindingligand is a proteinaceous enzyme substrate or inhibitor, there may beadditional amino acids, or an alkyl group, etc., between the metal ionligand and the functional substrate or inhibitor.

In addition, as noted herein, two or more binding ligands may beattached to a single redox active complex. For example, twosingle-stranded nucleic acids may be attached, such that the binding ofa complementary target sequence will change the solvent reorganizationenergy of the redox active molecule. In this embodiment, the two singlestranded nucleic acids are designed to allow for a “gap” in thecomplementary sequence to accommodate the metal ion; this is generallyfrom 1 to 3 nucleotides.

In a preferred embodiment, the binding ligand and the redox activemolecule do not form a redox active complex, but rather are eachindividually attached to the electrode, generally via a spacer. In thisembodiment, it is the proximity of the redox active molecule to thetarget analyte bound to the binding ligand that results in a decrease ofthe solvent reorganization energy upon binding. Preferably, the solventaccessible redox active molecule is within 12Δ of some portion of thetarget analyte, with less than about 8 Δ being preferred and less thanabout 5 Δ being particularly preferred, and less than about 3.5Δ beingespecially preferred. It should be noted that the distance between thebinding ligand and the redox active molecule may be much larger,depending on the size of the target analyte. Thus, the binding of alarge target analyte may reduce the solvent reorganization energy of asolvent accessible redox active molecule many angstroms away from thebinding ligand. A representative composition is depicted below inStructure 8:

In Structure 8, the binding ligand and the transition metal complex areseparately attached to the electrode. While Structure 8 depicts a 1:1ratio of transition metal complexes to binding ligands, this is notrequired; in fact, it may be preferable to have an excess of transitionmetal complexes on the electrode, particularly when the target analyteis relatively large in comparison to the transition metal complex. Thus,for example, a single binding event of a target analyte to a bindingligand can result in a decrease in solvent reorganization energy for anumber of transition metal complexes, if the density of the transitionmetal complexes is high enough in the area of the binding ligand, or thetarget analyte is large enough. Similarly, different binding ligands forthe same target analyte may be used; for example, to “tack down” a largetarget analyte on the surface, to effect as many transition metalcomplexes as possible per single target analyte.

The redox moieties and binding ligands are attached to an electrode, viaa spacer as outlined above. Thus, one end or terminus of the conductiveoligomer is attached to the redox moiety or binding ligand, and theother is attached to an electrode. In some embodiments it may bedesirable to have the conductive oligomer attached at a position otherthan a terminus, or to have a branched conductive oligomer that isattached to an electrode at one terminus and to a redox active moleculeand a binding ligand at other termini. Similarly, the conductiveoligomer may be attached at two sites to the electrode.

By “electrode” herein is meant a composition, which, when connected toan electronic device, is able to sense a current or charge and convertit to a signal. Preferred electrodes are known in the art and include,but are not limited to, certain metals and their oxides, including gold;platinum; palladium; silicon; aluminum; metal oxide electrodes includingplatinum oxide, titanium oxide, tin oxide, indium tin oxide, palladiumoxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo₂O₆), tungstenoxide (WO₃) and ruthenium oxides; and carbon (including glassy carbonelectrodes, graphite and carbon paste). Preferred electrodes includegold, silicon, carbon and metal oxide electrodes.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays are made,thus requiring addressable locations for both synthesis and detection.Alternatively, for single analyte analysis, the electrode may be in theform of a tube, with the compositions of the invention bound to theinner surface. This allows a maximum of surface area containing thebinding ligand to be exposed to a small volume of sample.

The covalent attachment of the conductive oligomer containing the redoxactive moieties and binding ligands of the invention may be accomplishedin a variety of ways, depending on the electrode and the conductiveoligomer used. Generally, some type of linker is used. For example, inStructure 6, F₁ may be a linker or atom. The choice of “F₁” will dependin part on the characteristics of the electrode. Thus, for example, F₁may be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, F₁ may be a silicon (silane)moiety attached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, F₁ may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10: 1306-1313 (1994)). Thus, preferred F₁ moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the conductive oligomer maybe attached to the electrode with more than one F₁ moiety; the F₁moieties may be the same or different. Thus, for example, when theelectrode is a gold electrode, and F₁ is a sulfur atom or moiety,multiple sulfur atoms may be used to attach the conductive oligomer tothe electrode. Preferably, the F₁ moiety is just a sulfur atom, butsubstituted sulfur moieties may also be used.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theF₁ moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention.

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup.

In general, one of two general schemes may be followed to synthesize thecompositions of the invention. In a preferred embodiment, the spacer issynthesized and the redox active complex, comprising the redox activemolecule and the binding ligand is also made separately. These two areadded together, and then added to the electrode. Alternatively, in apreferred embodiment, the spacer is made and attached to the electrode.The redox active complex is made, and then it is added to the spacer.General synthetic schemes may be found in PCT US97/20014.

Thus, in a preferred embodiment, electrodes are made that compriseconductive oligomers attached to redox active moieties and/or bindingligands for the purposes of analyte assays, as is more fully describedherein. As will be appreciated by those in the art, electrodes can bemade that have a single species of binding ligand (i.e. specific for aparticular analyte) or multiple binding ligand species (i.e. multipleanalytes).

In addition, as outlined herein, the use of a solid support such as anelectrode enables the use of these binding ligands in an array form. Theuse of arrays of binding ligands specific for oligonucleotides are wellknown in the art. In addition, techniques are known for “addressing”locations within an electrode and for the surface modification ofelectrodes.

Thus, in a preferred embodiment, arrays of different binding ligands arelaid down on the electrode, each of which are covalently attached to theelectrode via a conductive linker. In this embodiment, the number ofdifferent species of binding ligands may vary widely, from one tothousands, with from about 4 to about 100,000 being preferred, and fromabout 10 to about 10,000 being particularly preferred.

In a preferred embodiment, the electrode further comprises a passivationagent, preferably in the form of a monolayer on the electrode surface.For some analytes, such as nucleic acids, the efficiency of analytebinding (i.e. hybridization) may increase when the binding ligand is ata distance from the electrode. In addition, the presence of a monolayercan decrease non-specific binding to the surface. A passivation agentlayer facilitates the maintenance of the binding ligand and/or analyteaway from the electrode surface. In addition, a passivation agent servesto keep charge carriers away from the surface of the electrode. Thus,this layer helps to prevent electrical contact between the electrodesand the electron transfer moieties, or between the electrode and chargedspecies within the solvent. Such contact can result in a direct “shortcircuit” or an indirect short circuit via charged species which may bepresent in the sample. Accordingly, the monolayer of passivation agentsis preferably tightly packed in a uniform layer on the electrodesurface, such that a minimum of “holes” exist. Alternatively, thepassivation agent may not be in the form of a monolayer, but may bepresent to help the packing of the conductive oligomers or othercharacteristics.

The passivation agents thus serve as a physical barrier to block solventaccessibility to the electrode. As such, the passivation agentsthemselves may in fact be either (1) conducting or (2) nonconducting,i.e. insulating, molecules. Thus, in one embodiment, the passivationagents are conductive oligomers, as described herein, with or without aterminal group to block or decrease the transfer of charge to theelectrode. Other passivation agents which may be conductive includeoligomers of —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. In a preferredembodiment, the passivation agents are insulator moieties.

An “insulator” is a substantially nonconducting oligomer, preferablylinear. By “substantially nonconducting” herein is meant that the rateof electron transfer through the insulator is slower than the rate ofelectron transfer through the a conductive oligomer. Stated differently,the electrical resistance of the insulator is higher than the electricalresistance of the conductive oligomer. It should be noted however thateven oligomers generally considered to be insulators, such as —(CH₂)₁₆molecules, still may transfer electrons, albeit at a slow rate.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷Ω⁻¹ cm⁻¹ or lower, with less than about 10⁻⁸Ω⁻¹ cm⁻¹ beingpreferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

The passivation agents, including insulators, may be substituted with Rgroups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. In addition, the terminus ofthe passivation agent, including insulators, may contain an additionalgroup to influence the exposed surface of the monolayer. For example,the addition of charged, neutral or hydrophobic groups may be done toinhibit non-specific binding from the sample, or to influence thekinetics of binding of the analyte, etc. For example, there may benegatively charged groups on the terminus to form a charged surface suchthat when the nucleic acid is DNA or RNA the nucleic acid is repelled orprevented from lying down on the surface.

The length of the passivation agent will vary as needed. Generally, thelength of the passivation agents is similar to the length of theconductive oligomers, as outlined above. In addition, the conductiveoligomers may be basically the same length as the passivation agents orlonger than them, resulting in the binding ligands being more accessibleto the solvent.

The monolayer may comprise a single type of passivation agent, includinginsulators, or different types.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

The passivation agents are generally attached to the electrode in thesame manner as the conductive oligomer, and may use the same “F₁” linkeras defined above.

The target analyte, contained within a test sample, is added to theelectrode containing either a solvent accessible redox active complex ora mixture of solvent accessible transition metal complexes and bindingligands, under conditions that if present, the target analyte will bindto the binding ligand. These conditions are generally physiologicalconditions. Generally a plurality of assay mixtures are run in parallelwith different concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration or below the level ofdetection. In addition, any variety of other reagents may be included inthe screening assay. These include reagents like salts, neutralproteins, e.g. albumin, detergents, etc which may be used to facilitateoptimal binding and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used. The mixture of components may be added in any orderthat provides for the requisite binding.

In a preferred embodiment, the target analyte will bind the bindingligand reversibly, i.e. non-covalently, such as, in protein-proteininteractions of antigens-antibodies, enzyme-substrate (or someinhibitors) or receptor-ligand interactions.

In a preferred embodiment, the target analyte will bind the bindingligand irreversibly, for example covalently. For example, someenzyme-inhibitor interactions are considered irreversible.Alternatively, the analyte initially binds reversibly, with subsequentmanipulation of the system which results in covalent attachment. Forexample, chemical cross-linking after binding may be done, as will beappreciated by those in the art. For example, peptides may becross-linked using a variety of bifunctional agents, such asmaleimidobenzoic acid, methyldithioacetic acid, mercaptobenzoic acid,S-pyridyl dithiopropionate, etc. Alternatively, functionally reactivegroups on the target analyte and the binding ligand may be induced toform covalent attachments.

Upon binding of the analyte to the binding moiety, the solventaccessible transition metal complex becomes solvent inhibited. By“solvent inhibited transition metal complex” herein is meant the solventreorganization energy of the solvent inhibited transition metal complexis less than the solvent reorganization energy of the solvent accessibletransition metal complex. As noted above, this may occur in severalways. In a preferred embodiment, the target analyte provides acoordination atom, such that the solvent accessible transition metalcomplex loses at least one, and preferably several, of its small polarligands. Alternatively, in a preferred embodiment, the proximity of thetarget analyte to the transition metal complex does not result in ligandexchange, but rather excludes solvent from the area surrounding themetal ion (i.e. the first or second coordination sphere) thuseffectively lowering the required solvent reorganization energy.

In a preferred embodiment, the required solvent reorganization energydecreases sufficiently to result in a decrease in the E⁰ of the redoxactive molecule by at about 100 mV, with at least about 200 mV beingpreferred, and at least about 300-500 mV being particularly preferred.

In a preferred embodiment, the required solvent reorganization energydecreases by at least 100 mV, with at least about 200 mV beingpreferred, and at least about 300-500 mV being particularly preferred.

In a preferred embodiment, the required solvent reorganization energydecreases sufficiently to result in a rate change of electron transfer(k_(ET)) between the solvent inhibited transition metal complex and theelectrode relative to the rate of electron transfer between the solventaccessible transition metal complex and the electrode. In a preferredembodiment, this rate change is greater than about a factor of 3, withat least about a factor of 10 being preferred and at least about afactor of 100 or more being particularly preferred.

The determination of solvent reorganization energy will be done as isappreciated by those in the art. Briefly, as outlined in Marcus theory,the electron transfer rates (k_(ET)) are determined at a number ofdifferent driving forces (or free energy, −ΔA

); the point at with the rate equals the free energy is theactivationless rate (λ). This may be treated in most cases as theequivalent of the solvent reorganization energy; see Gray et al. Ann.Rev. Biochem. 65:537 (1996), hereby incorporated by reference.

The solvent inhibited transition metal complex, indicating the presenceof a target analyte, is detected by intiating electron transfer anddetecting a signal characteristic of electron transfer between thesolvent inhibited redox active molecule and the electrode.

Electron transfer is generally initiated electronically, with voltagebeing preferred. A potential is applied to a sample containing modifiednucleic acid probes. Precise control and variations in the appliedpotential can be via a potentiostat and either a three electrode system(one reference, one sample and one counter electrode) or a two electrodesystem (one sample and one counter electrode). This allows matching ofapplied potential to peak electron transfer potential of the systemwhich depends in part on the choice of transition metal complexes and inpart on the conductive oligomer used.

Preferably, initiation and detection is chosen to maximize the relativedifference between the solvent reorganization energies of the solventaccessible and solvent inhibited transition metal complexes.

Electron transfer between the transition metal complex and the electrodecan be detected in a variety of ways, with electronic detection,including, but not limited to, amperometry, voltammetry, capacitance andimpedance being preferred. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, and filtering (high pass, low pass, band pass). In someembodiments, all that is required is electron transfer detection; inothers, the rate of electron transfer may be determined.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedance. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry, and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between theelectrode containing the compositions of the invention and an auxiliary(counter) electrode in the test sample. Electron transfer of differingefficiencies is induced in samples in the presence or absence of targetanalyte.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the redox active molecule.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the redox active molecules and theelectrode. In addition, other properties of insulators (such asresistance) and of conductors (such as conductivity, impedance andcapacitance) could be used to monitor electron transfer between theredox active molecules and the electrode. Finally, any system thatgenerates a current (such as electron transfer) also generates a smallmagnetic field, which may be monitored in some embodiments.

In a preferred embodiment, the system may be calibrated to determine theamount of solvent accessible transition metal complexes on an electrodeby running the system in organic solvent prior to the addition oftarget. This is quite significant to serve as an internal control of thesensor or system. This allows a preliminary measurement, prior to theaddition of target, on the same molecules that will be used fordetection, rather than rely on a similar but different control system.Thus, the actual molecules that will be used for the detection can bequantified prior to any experiment. Running the system in the absence ofwater, i.e. in organic solvent such as acetonitrile, will exclude thewater and substantially negate any solvent reorganization effects. Thiswill allow a quantification of the actual number of molecules that areon the surface of the electrode. The sample can then be added, an outputsignal determined, and the ratio of bound/unbound molecules determined.This is a significant advantage over prior methods.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on electronic current. The fast rates of electron transfer of thepresent invention result both in high signals and stereotyped delaysbetween electron transfer initiation and completion. By amplifyingsignals of particular delays, such as through the use of pulsedinitiation of electron transfer and “lock-in” amplifiers of detection,orders of magnitude improvements in signal-to-noise may be achieved.

Without being bound by theory, it appears that target analytes, bound toan electrode, may respond in a manner similar to a resistor andcapacitor in series. Also, the E⁰ of the redox active molecule can shiftas a result of the target analyte binding. Furthermore, it may bepossible to distinguish between solvent accessible and solvent inhibitedtransition metal complexes on the basis of the rate of electrontransfer, which in turn can be exploited in a number of ways fordetection of the target analyte. Thus, as will be appreciated by thosein the art, any number of initiation-detection systems can be used inthe present invention.

In a preferred embodiment, electron transfer is initiated and detectedusing direct current (DC) techniques. As noted above, the E⁰ of theredox active molecule can shift as a result of the change in the solventreorganization energy upon target analyte binding. Thus, measurementstaken at the E⁰ of the solvent accessible transition metal complex andat the E⁰ of the solvent inhibited complex will allow the detection ofthe analyte. As will be appreciated by those in the art, a number ofsuitable methods may be used to detect the electron transfer.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. A first input electrical signal isapplied to the system, preferably via at least the sample electrode(containing the complexes of the invention) and the counter electrode,to initiate electron transfer between the electrode and the secondelectron transfer moiety. Three electrode systems may also be used, withthe voltage applied to the reference and working electrodes. In thisembodiment, the first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1 V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 10 MHz, with from about 1Hz to about 1 MHz being preferred, and from about 1 Hz to about 100 kHzbeing especially preferred.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the electron transfer moiety. The sweep is used to identifythe DC voltage at which the maximum response of the system is seen. Thisis generally at or about the electrochemical potential of the transitionmetal complex. Once this voltage is determined, either a sweep or one ormore uniform DC offset voltages may be used. DC offset voltages of fromabout −1 V to about +1.1 V are preferred, with from about −500 mV toabout +800 mV being especially preferred, and from about −300 mV toabout 500 mV being particularly preferred. On top of the DC offsetvoltage, an AC signal component of variable amplitude and frequency isapplied. If the transition metal complex has a low enough solventreorganization energy to respond to the AC perturbation, an AC currentwill be produced due to electron transfer between the electrode and thetransition metal complex.

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, as noted above, it may be possible todistinguish between solvent accessible and solvent inhibited transitionmetal complexes on the basis of the rate of electron transfer, which inturn can be used either to distinguish the two on the basis of frequencyor overpotential.

In a preferred embodiment, measurements of the system are taken at leasttwo separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe transition metal complexes, higher frequencies result in a loss ordecrease of output signal. At some point, the frequency will be greaterthan the rate of electron transfer through even solvent inhibitedtransition metal complexes, and then the output signal will also drop.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe target analyte, i.e. “locking out” or “filtering” unwanted signals.That is, the frequency response of a charge carrier or redox activespecies in solution will be limited by its diffusion coefficient.Accordingly, at high frequencies, a charge carrier may not diffuserapidly enough to transfer its charge to the electrode, and/or thecharge transfer kinetics may not be fast enough. This is particularlysignificant in embodiments that do not utilize a passivation layermonolayer or have partial or insufficient monolayers, i.e. where thesolvent is accessible to the electrode. As outlined above, in DCtechniques, the presence of “holes” where the electrode is accessible tothe solvent can result in solvent charge carriers “short circuiting” thesystem. However, using the present AC techniques, one or morefrequencies can be chosen that prevent a frequency response of one ormore charge carriers in solution, whether or not a monolayer is present.This is particularly significant since many biological fluids such asblood contain significant amounts of redox active species which caninterfere with amperometric detection methods.

In a preferred embodiment, measurements of the system are taken at leasttwo separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.In a preferred embodiment, the frequency response is determined at leasttwo, preferably at least about five, and more preferably at least aboutten frequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on the overpotential/amplitude of the inputsignal; the frequency of the input AC signal; the composition of theintervening medium, i.e. the impedance, between the electron transfermoieties; the DC offset; the environment of the system; and the solvent.At a given input signal, the presence and magnitude of the output signalwill depend in general on the solvent reorganization energy required tobring about a change in the oxidation state of the metal ion. Thus, upontransmitting the input signal, comprising an AC component and a DCoffset, electrons are transferred between the electrode and thetransition metal complex, when the solvent reorganization energy is lowenough, the frequency is in range, and the amplitude is sufficient,resulting in an output signal.

In a preferred embodiment, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In addition, those in the art will appreciate that it is also possibleto use the compositions of the invention in assays that rely on a lossof signal. For example, a first measurement is taken when the transitionmetal complex is inhibited, and then the system is changed as a resultof the introduction of a target analyte, causing the solvent inhibitedmolecule to become solvent accessible, resulting in a loss of signal.This may be done in several ways, as will be appreciated by those in theart.

In a preferred embodiment, a first measurement is taken when the targetanalyte is present. The target analyte is then removed, for example bythe use of high salt concentrations or thermal conditions, and then asecond measurement is taken. The quantification of the loss of thesignal can serve as the basis of the assay.

Alternatively, the target analyte may be an enzyme. In this preferredembodiment, the transition metal complex is made solvent inhibited bythe presence of an enzyme substrate or analog, preferably, but notrequired to be covalently attached to the transition metal complex,preferably as one or more ligands. Upon introduction of the targetenzyme, the enzyme associates with the substrate to cleave or otherwisesterically alter the substrate such that the transition metal complex ismade solvent accessible. This change can then be detected. Thisembodiment is advantageous in that it results in an amplification of thesignal, since a single enzyme molecule can result in multiple solventaccessible molecules. This may find particular use in the detection ofbacteria or other pathogens that secrete enzymes, particularly scavengerproteases or carbohydrases.

Similarly, a preferred embodiment utilizes competition-type assays. Inthis embodiment, the binding ligand is the same as the actual moleculefor which detection is desired; that is, the binding ligand is actuallythe target analyte or an analog. A binding partner of the binding ligandis added to the surface, such that the transition metal complex becomessolvent inhibited, electron transfer occurs and a signal is generated.Then the actual test sample, containing the same or similar targetanalyte which is bound to the electrode, is added. The test sampleanalyte will compete for the binding partner, causing the loss of thebinding partner on the surface and a resulting decrease in the signal.

A similar embodiment utilizes a target analyte (or analog) is covalentlyattached to a preferably larger moiety (a “blocking moiety”). Theanalyte-blocking moiety complex is bound to a binding ligand that bindsthe target analyte, serving to render the transition metal complexsolvent inhibited. The introduction of the test sample target analyteserves to compete for the analyte-blocking moiety complex, releasing thelarger complex and resulting in a more solvent accessible molecule.

In addition, while the majority of the above discussion is directed tothe use of the invention when the compositions are attached to surfacessuch as electrodes, those of skill in the art will appreciate thatsolution-based systems are also possible. In this embodiment, solventaccessible transition metal complexes are attached to binding ligands(either directly or using short linkers that keep the binding ligand andthe transition metal complex in close enough proximity to allowdetection) to form soluble redox active complexes. Upon binding of ananalyte, the transition metal complex becomes solvent inhibited, and achange in the system can be detected. In a preferred embodiment, thereaction is monitored by fluorescence or electrochemical means.Alternatively, the reaction may be monitored electronically, usingmediators.

The present invention further provides apparatus for the detection ofanalytes using AC detection methods. The apparatus includes a testchamber which has at least a first measuring or sample electrode, and asecond measuring or counter electrode. Three electrode systems are alsouseful. The first and second measuring electrodes are in contact with atest sample receiving region, such that in the presence of a liquid testsample, the two electrodes may be in electrical contact.

In a preferred embodiment, the first measuring electrode comprises aredox active complex, covalently attached via a spacer, and preferablyvia a conductive oligomer, such as are described herein. Alternatively,the first measuring electrode comprises covalently attached transitionmetal complexes and binding ligands.

The apparatus further comprises a voltage source electrically connectedto the test chamber; that is, to the measuring electrodes. Preferably,the voltage source is capable of delivering AC and DC voltages, ifneeded.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target analyte.

The compositions of the present invention may be used in a variety ofresearch, clinical, quality control, or field testing settings.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLE Preparation of Solvent Accessible Redox Active Moiety

In this example, a solution based sensor was made, using a redox activecomplex comprising a ruthenium complex and a biotin binding ligand. Atransition metal complex of ruthenium, with small polar coordinationligands (NH₃), was made. One of the coordination atoms was provided bythe binding ligand norbiotin (biotin conjugated with a primary amine viafour carbon linker), such that upon binding of avidin, the transitionmetal complex goes from solvent accessible to solvent inhibited. Thiswas detected fluorometrically. Alternatively, the redox active complexof ruthenium and biotin can be activated and added to a surface to forman electrode-based sensor.

The synthesis of [trans RuIII (NH₃)₄(norbiotin) Cl]C₁₋₂ was carried inseveral steps. The first intermediate in the reaction sequence istrans-[SO₂ (NH₃) RuIICl]Cl] and was synthesized in the following manner.

2.5 gr. (8.5 mmoles) of [(NH₃)₅ RuIIICl]Cl₂ was slurried in 65 ml ofpre-heated water (˜70° C.) in a three neck round bottom flask equippedwith a thermometer, reflux condenser and gas inlet. To this flask wasadded 3.55 gr (2.4 mmoles) of NaHSO₃ and immediately a continuous streamof SO gas was bubbled through the solution and the mixture allowed towarm to 83° C. The reaction was allowed to proceed for 90 minutes. Thesolution was cooled to 0° C., and the product collected and washedseveral times with acetone.

The solid was slurried in 200 mls of 6M HCl and heated to a vigorousreflux for 20 min in a 500 ml flask. The reaction mixture was filteredand allowed to stand at 4° C. overnight. The rust colored crystals oftrans-[SO(NH₃) RuIICl]Cl were collected and slurried in 50 ml of water,heated to 40° C., an excess of norbiotin was added and the solutionallowed to react for 30 minutes. The solution was transferred to a 1000ml flask and 750 ml of acetone was added and allowed to stir for 10minutes. The solid was collected, washed with acetone and dried invacuo.

The solid was dissolved in a minimum of water and filtered. To thissolution was added dropwise with stirring a 50:50 mixture of 30% H₂O₂and 2N HCl. A solid was obtained by the addition of 15 volumes ofacetone, collected and dried in vacuo. This product was dissolved in aminimum amount of degassed 0.15N HCl and thoroughly degassed. Zinc-Hgamalgam was prepared, the solution was transferred to the zinc amalgam,and the reaction allowed to proceed for 1 hour. A previously degassedsolution of 1M BaCl₂ was added.

The solid, including the amalgam, was filtered as quickly as possibleinto a filter flask containing 3-4 ml of 30% H₂O₂ and 3M HCl. Theproduct was obtained from the yellow solution via precipitation using 15volumes of acetone, collected, washed and dried in vacuo. The solid wasredissolved in a minimum amount of 0.01N HCl and applied to a 4×30 cmcolumn of SP Sephadex C-25. The product was recovered using 0.2N HCl.

The collected fractions were evaporated to dryness, dissolved in aminimum amount of 0.01N HCl, filtered and precipitated with 15 volumesof acetone. The product [trans RuIII (NH₃)₄(norbiotin) Cl]Cl₂ wascollected, and dried in vacuo.

For a solution-based sensor, the material was taken up in water and afluorescence measurement was taken; the sample exhibited nofluorescence; that is, the presence of water was a barrier tofluorescence. Avidin was added and a second fluorescence measurement wastaken; in the presence of avidin, fluorescence was detected. This showsthat the environment around the complex is altered such that the wateris no longer a barrier to fluorescence; i.e. fluorescence is notquenched.

For an electrode-based sensor, the material can be activated foraddition to a conductive oligomer as follows. The [trans RuIII(NH₃)₄(norbiotin) Cl]Cl₂ is activated by reduction of the complex usingZinc-Hg amalgam to form [trans RuII (NH₃)₄(norbiotin) H₂O]Cl₂ underinert atmosphere conditions. To this material is added a conductiveoligomer that terminates in a group suitable to serve as a coordinationatom, such as a nitrogen-containing species, such as analine. Theconductive oligomer containing the redox active complex (i.e. thesolvent accessible transition metal complex and the binding ligand) canthen be mixed with other monolayer-forming components such aspassivation agents and added to the electrode using known techniques,such as those described in PCT US97/20014, hereby incorporated byreference.

1. A method of detecting a target analyte in a test sample comprising a)adding a target analyte to an electrode comprising i) a solventaccessible transition metal complex comprising a metal selected from thegroup consisting of manganese, technetium, rhenium, iron, ruthenium,osmium, cobalt, rhodium, iridium, nickel, palladium, platinum, copper,silver and gold; and ii) a binding ligand that will bind said targetanalyte; such that upon binding of said target analyte to said bindingligand, a solvent inhibited transition metal complex is formed; and b)measuring a change in E⁰ of said transition metal complex.
 2. A methodaccording to claim 1, wherein said measuring comprises applying at leasta first input signal to said solvent inhibited transition metal complexand receiving an output signal.
 3. A method according to claim 2,wherein in the absence of target analyte, said first input signal doesnot result in a significant change in E⁰.
 4. A method according to claim2, wherein said first input signal comprises at least an AC component.5. A method according to claim 2, further comprising applying inputsignal at a plurality of frequencies.
 6. A method according to claim 2or 4, wherein said input signal comprises at least a DC component.
 7. Amethod according to claim 6, further comprising applying input signal ata plurality of voltages.
 8. A method according to claim 2, wherein saidoutput signal is a current.
 9. A method according to claim 8, whereinsaid current is an AC current.
 10. A method according to claim 1 whereinsaid electrode further comprises gold.
 11. A method according to claim 1wherein said electrode further comprises graphite-pyrene.
 12. A methodaccording to claim 1 wherein said electrode further comprises aself-assembled monolayer.
 13. A method according to claim 12 whereinsaid self assembled monolayer comprises conductive oligomers.
 14. Amethod according to claim 1, wherein said solvent accessible transitionmetal complex has at least one coordination site occupied by a polarcoordination group.
 15. A method according to claim 1, wherein saidsolvent accessible transition metal complex has at least onecoordination site occupied by a water molecule.
 16. A method accordingto claim 1, wherein said solvent accessible transition metal complex hasat least two coordination sites occupied by a polar coordination group.17. A method according to claim 1, wherein said binding ligand iscovalently attached to said solvent accessible transition metal complex.18. A method according to claim 1, wherein said binding ligand iscovalently attached to said electrode.
 19. A method according to claim1, wherein said solvent accessible transition metal complex iscovalently attached to said electrode.
 20. A method according to claim18, wherein said covalent attachment is via a spacer.
 21. A methodaccording to claim 19, wherein said covalent attachment is via a spacer.22. A method according to claim 20 or 21, wherein said spacer is aconductive oligomer.